DNA detection by means of a strand reassociation complex

ABSTRACT

The discriminating capability of hybridization assays is increased by a combination of labelled primers which produce amplificates of one strand of a nucleic acid with a capture probe which is complementary to the same strand of the nucleic acid.

This is a Continuation of application Ser. No. 09/325,554 filed Jun. 4,1999, now U.S. Pat. No. 6,410,235. The disclosure of the priorapplication(s) is hereby incorporated by reference herein in itsentirety.

The invention concerns a method for the detection of nucleic acids usingthe amplification of the nucleic acid with the aid of labelled primersand detection of the amplificate with the aid of a capture probe.

Biospecific binding assays which enable the detection of certainanalytes or analyte characteristics by means of molecular recognitionmechanisms have become indispensable in diagnostics and bioanalytics. Inthis connection hybridization assays have become firmly established inrecent years in addition to immunoassays and receptor ligand assays.Hybridization assays utilize the principle of nucleobase pairing (A::T;G:::C) for the molecular recognition of certain analyte nucleic acids(e.g. DNA, RNA) by probes with the desired specificity. Thus for exampleoligonucleotide probes which are composed of 18-20 nucleotides in achosen sequence enable unequivocal detection even over the entire humangenome.

Hybridization assays (=probe based assays) have been given aninteresting and promising extension by so-called NA chip technologies.In these at least 2 and usually several to very many probes withdifferent sequences and thus different specificity are bound in ageometric pattern in separate areas on a test carrier so that acorresponding number of hybridization reactions between the probes andnucleic acid analyte segments or different nucleic acid analytes can becarried out concurrently. Under suitable reaction conditions e.g.sequence selection, buffer environment, salt content and above all theincubation and wash temperature, it is possible to keep only thosehybridization complexes bound to the solid phase in which all thenucleotides contained in the oligonucleotide probe are complementary tothe corresponding nucleotides in the analyte molecule resulting in thefull binding strength. This is then referred to as complete base pairing(perfect match, PM).

Hybridization complexes which contain mismatches (MM) are detached undersuch conditions. Under optimal conditions it is even possible tounequivocally distinguish between complexes with complete base pairingsand complexes with 1-point mismatches (single base transitions). Sincethis occurs concurrently on the solid phase when using a geometricpattern of capture probes (array), it is referred to as probe arraytesting.

The capture probes can all have a constant length (number of nucleotidebuilding blocks) or the oligo length can be inversely proportionallymatched to the GC content. In the first case a common meltingtemperature Tm can be achieved for all completely paired hybridizationcomplexes by buffer additives which for example strengthen AT bonds tosuch an extent that the Tm is independent of the nucleobase sequence andis only dependent on the oligo length. Examples of such additives aretetramethylammonium chloride (TMAC) and tetraethylammonium bromide. Inthe second case the stated length adaptation results in a Tm levelling.The capture probes can have chemically different backbones which carrythe specificity-mediating nucleobases e.g. deoxyribosyl-phosphodiesterstrands (=>DNA), ribosyl-phosphodiester strands (=>RNA) or they canbelong to a non-natural class of substances e.g. N-(2-aminoethyl)-glycylor N-(2-aminoethyl)glutamyl strands (=>PNA, WO 92/20702).

Probe array testing is of interest for many molecular biological ordiagnostic applications. These include multipathogen testing(simultaneous detection of different pathogens on a gene level),(sub)typing of organisms, analysis of genetic diversity (polymorphisms,mutations), sequencing of genes or genomes etc.

Nucleic acids are relatively complex analytes which usually have to befirstly isolated, then amplified and, in the case of DNA, renderedsingle-stranded (denatured) before they can be used in a probe basedassay or probe array testing. This processing and the fact thatcomplementary nucleobases also have a tendency for base pairing withinone and the same strand result in some typical difficulties such as avariable analyte titre in the reaction solution due to variations in theefficiency of the isolation or amplification, a suboptimal denaturingefficiency, reassociation of the single strands of a DNA to form theoriginal double strand which competes with the hybridization of a singlestrand with a probe, internal strand hybridization (formation ofsecondary structures e.g. hairpin loops or cross formations) whichcompete with the probe hybridization. This becomes more pronounced asthe palindrome index increases i.e. the degree of self-complementarityof a DNA or RNA strand.

Especially the last two phenomena essentially determine theaccessibility of the sequence region of the analyte which is the basisfor the test and hence limit the overall performance of an entire arrayof capture probes.

The so-called PCR method (polymerase chain reaction, U.S. Pat. No.4,683,202) is usually used to amplify the analyte nucleic acid. In thismethod it is possible to already incorporate a detectable group duringthe amplification e.g. a digoxigenin derivative (DIG-labelling, EP-B-0324 474). This can be achieved by replacing a part of the dTTP byDIG-dUTP in the nucleoside triphosphate mixture.

A method is described in DE-A-3807994 (U.S. Pat. No. 5,476,769) in whichdetectably-labelled amplicon strands are hybridized to an immobilizablecapture probe and the hybrids that are formed are detected.

The so-called sandwich hybridization method is described in EP-A-0 079139 in which a nucleic acid to be detected is detected by hybridizationwith a capture probe and a detection probe which are complementary todifferent regions of the nucleic acid.

The object of the present invention was to improve hybridization assayswhich are based on a capture reaction of amplificates that have beengenerated while incorporating a label, in particular with regard totheir discrimination between two or more nucleic acids with very closelyrelated sequences.

A subject matter of the present invention is a method for the selectivedetection of a nucleic acid comprising the steps amplification of thenucleic acid or of a part thereof with the aid of two primers one ofwhich can hybridize with one strand of the nucleic acid to be detectedand the other can hybridize with a complementary strand thereto at leastone of which contains a bound detectable label and to form each time anextension product of these primers in a reaction mixture, binding acapture probe to one of these extension products to form a hybridizationcomplex which contains the capture probe and at least this extensionproduct, separating the extension product bound to the capture probefrom non-bound components of the reaction mixture and determining thedetectable label bound to the capture probe wherein the capture probe isselected such that it can bind with the strand of the extension productwhich can also hybridize with the extension product formed by extensionof the labelled primer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically the individual components for amplificationof the nucleic acid or a part thereof according to the invention.

FIG. 2 shows schematically a complex comprising strand T1, strand T2 andcapture probe P bound to a solid phase.

FIG. 3 shows the position of several regions with mutations in a rpoβ-gene segment.

FIG. 4 shows a folding proposal for a rpo β-gene segment.

FIG. 5 compares the real situation A (folded DNA with internal strandassociation) and the ideal situation B (DNA with no internal strandassociation).

The nomenclature of the individual components of the amplification asdefined by the invention are shown schematically in FIG. 1. The meaningsare as follows:

P1 forward primer P2 reverse primer T1 sense daughter strand (senseextension product) T2 anti-sense daughter strand (anti-sense extensionproduct) M1 anti-sense template strand (template); strand of the nucleicacid to be detected M2 sense template strand (template); opposite strandto the nucleic acid to be detected

A complex that is already bound to the solid phase comprising thestrands T1 (unlabelled), T2 (5′-terminally labelled) and the solidphase-bound probe P is shown schematically in FIG. 2. The rectangle is adetectable label.

The position of the regions with mutations in the segment is shown inFIG. 3.

A folding proposal for the rpo β-gene segment is shown in FIG. 4.

FIG. 5 shows a comparison between the ideal situation (rod-shaped DNA)and the real situation A (folded). It is clear that the accessibility ofthe terminal label is in fact greatly impaired. This impairment isreduced or avoided by the procedure of the present invention.

The core of the present invention is the surprising observation that thebest discrimination power (differentiation between PM and MM) isachieved when the capture probes bound to the solid phase are designedto be complementary and anti-parallel to the sense strand which issynthesized in the PCR starting from an unlabelled forward primer, butthe detection reaction is based on the anti-sense strand which issynthesized in the PCR starting from a reverse primer labelled with adetectable group so that the probe hybridization complex whichdetermines the specificity (discriminating power) is detected indirectlyand optionally quantified via the DNA strand reassociation complex.Conversely the capture probe can also be complementary to the anti-sensestrand and detention is by means of reassociation with the sense strandthat can be detected by the labelled forward primer.

Although this also has an effect on single hybridizations, it has aparticularly positive effect in connection with hybridization assayscarried out concurrently i.e. (D)NA probe array testing.

A selective test is understood as a test which selects and binds anucleic acid with a target sequence that only differs slightly from anon-target sequence in a sample containing numerous nucleic acids thatdo not have the target sequence and among which the target sequence maybe present. The binding occurs by pairing of complementary bases, suchas A and T or U as well as G and C, of a probe and of the nucleic acidto be detected. For this purpose the sequence of the probe is highlycomplementary, preferably 100% complementary, to the target sequence ofthe nucleic acid especially with regard to those bases where the targetsequence differs from the non-target sequences. The probe sequence ispreferably between 8 and 36 nt, particularly preferably between 16 and20 nt long. The chemical structure of the probe especially in thenon-nucleobase part, e.g. the so-called backbone, can be relativelyindependently selected provided selective binding to the target sequenceis still possible. In addition to the (preferred) oligonucleotides (thenatural backbone or a sugar phosphate backbone modified by attachinggroups serves as the backbone), the so-called peptide nucleic acids (PNAaccording to WO 92/20702, artificial amino acids incorporated into thestructure of the backbone) have recently also proven to be suitable.Molecules with mixed backbone units are also suitable (WO 95/14706 orEP-A-0 672 700).

A capture probe whose function is to sequence-specifically immobilizethe reaction complexes on the solid phase can for example bind to thestrand of the extension product which can also hybridize with theextension product formed by extension of the labelled primer if it iscomplementary to a sequence on this extension product. This sequence isthen the target sequence. This sequence of the nucleic acid to bedetected is either known, can be determined by sequencing or is notdefined and determined until it has bound to one or several captureprobe(s) according to the invention.

The target sequence is preferably selected such that it is between theinner ends of the primers which face towards one another. The sequenceof the target sequence is selected such that it differs from sequencesof nucleic acids that are not to be detected especially with regard toits base sequence. The differences can for example be due to mutations(single or several base substitutions), deletions, insertions orpolymorphisms.

A person skilled in the art can for example find suitable targetsequences by sequence searches and sequence comparison in known sequencedata bases. In the spirit of a preferred embodiment of the invention inwhich several sequences, sequence differences, mutations, polymorphismsor the like are detected, the target sequences are preferably within aregion which can be amplified with the aid of a single primer pair (areverse and a forward primer). However, in principle, it is alsopossible to use several primers especially when they all lead toamplificates which contain, among others, the regions corresponding tothe other target sequences. The nucleic acid to be detected can be RNAor DNA of any desired origin. Genomic DNA is preferred.

A nucleic acid or sequence that is complementary to another nucleic acidor sequence is understood in this case as a nucleic acid or sequencewhich contains an uninterrupted, consecutive series of bases which canform Watson-Crick or/and Hoogsteen base pairings with an uninterrupted,consecutive series of bases of the other nucleic acid or sequence. Thisseries is preferably longer than 10 bases.

A nucleic acid or sequence which is homologous to another nucleic acidor sequence is understood in this case as a nucleic acid or sequencewhich is complementary to a nucleic acid or sequence which iscomplementary to the other nucleic acid. In this process uninterruptedsequences of bases are compared in each case.

In this connection it is obvious to a person skilled in the art thatthere are also artificial bases in addition to the naturally occurringbases which do not differ significantly from the natural bases withregard to their ability to bind to other bases. In the case ofoligonucleotides as probes this is also referred to as hybridization.The conditions under which an optimal hybridization occurs are familiarto a person skilled in the art. For example the temperature, probelength and salt content also depend on the GC content of the nucleicacid to be detected.

An advantage of the present method is that a higher selectivity of thedetection can be achieved under otherwise identical conditions. On theother hand, the stringency of the process parameters (e.g. washtemperature) can be lowered while retaining the selectivity. Selectivityis understood as specific probe hybridization to nucleic acids with anexactly complementary base sequence while discriminating against allnucleic acids with a sequence that is not exactly complementary.

Samples within the sense of the invention are liquids such as bodyfluids, culture media or tissue, or products prepared therefrom such aslysates, extracts or isolates such as serum, plasma or products derivedtherefrom.

Methods for amplification are basically known to a person skilled in theart. Examples are amplifications such as NASBA (EP-B-0 329 822 or U.S.Pat. No. 5,130,238), LCR (EP-B-0 320 208) and PCR (WO 90/01069).However, amplification by the PCR principle (EP-B-0 200 362 or U.S. Pat.No. 4,683,202) is preferred within the sense of the invention. In thismethod at least two primers are used whose sequence is selected suchthat one can hybridize with a strand of the nucleic acid to be detectedin such a way that, using this as a template and after the action of apolymerase and the other reagents necessary for this such as buffer,mononucleoside triphosphates etc, an extension product is formed using anucleic acid segment as a template that is located downstream of the 3′end of the primer which in turn can serve as a template forhybridization with and extension of the other primer. The moreselectively the primers bind to the nucleic acid or to its oppositestrand, the more selective the amplification becomes. Numerous copies ofthe nucleic acid piece located between the outer ends of the primerswhich contains the target sequence(s) can be prepared by many repeats ofthe reaction steps: hybridization of the primer with its template,extension of the primer and separation of the extension product from itstemplate.

A forward primer is understood as a primer which can or does hybridizewith a sequence of the nucleic acid strand to be detected. The strandcan be selected from one of the two strands of a double strand but itcan also already be a single-stranded nucleic acid e.g. RNA, preferablymRNA or rRNA. The reverse primer is a primer which can or does hybridizewith the extension product formed from the forward primer.

The forward primer is preferably a primer which can hybridize with theanti-sense strand of the nucleic acid to be detected, and the reverseprimer hybridizes with the sense strand. Hence a sense extension product(sense daughter strand) is formed from the forward primer, FIG. 1.

In addition to a sequence which targets the primer binding site, theprimers can also contain additional sequences which cannot directlyhybridize with the nucleic acid to be detected such as oligo T endswhich serve as spacers to a label preferably at the 5′ end. The captureprobes can also be modified in this manner.

Common primers have a length of between 12 and 30 nucleotides/bases. Inthe present invention these are both included in the subsequentdetection which is an indication for the presence (qualitative test) oramount (quantitative test) of the analyte nucleic acid.

In the sense of the invention at least one but preferably only one ofthe primers selected from the group of forward and reverse primers andparticularly preferably the reverse primer is detectably labelled. Alabel is understood as a chemical group whose properties differ in adetectable manner from those of nucleic acids. Such a property can forexample be an absorbance at a particular wavelength, fluorescence,scattering, reflection or ability to bind to other substances. One canalso divide them into direct and indirect labels. Direct labels can bedetected as a result of their signal-generating property without theaddition of further binding components. This group for example includesenzymes such as alkaline phosphatase or peroxidase (POD) which can bedetected by monitoring the enzyme-catalysed conversion of a coloursubstrate. However, groups which fulfil these conditions are onlypreferred within the sense of the invention when the substrate remainsin the vicinity of the complex during the measurement as is for examplethe case with aequorin. However, these also include signal-generatinggroups such as fluorescein which can be detected as a result of theirspectral properties.

Indirect labels are those which require binding to an additional reagentwhich contains a component that can bind to them which can then in turnbe directly or indirectly labelled. These groups include for examplehaptens such as biotin or digoxigenin. They can be detected by reactionwith a detection reagent containing their binding partner e.g.streptavidin or an antibody; for this purpose they are labelled with asignal-generating or signal-mediating group such as aequorin or afluorophore. Particulate components such as beads or microspheres madeof latex which contain a detectable dye are particularly preferred as asignal-generating group. The invention has a particularly positiveeffect with particles of a large size which cannot bind to the labelbecause of the steric situation shown in FIG. 5 (here the label isdigoxigenin) if internal strand reassociation is not prevented. Thelabel is of course expediently attached to the primer in such a way thatit hinders as little as possible the hybridization and extension of theprimer. In this connection it is preferable to attach it to the 5′ endin the case of an oligonucleotide e.g. during the chemical synthesisusing a phosphoramidite derivatized with the marker group. However, thelabel can also be attached to a base (WO 93/05060). In the case of PNAthe label is preferably attached to the amino end (WO 92/20703).

A capture probe is understood as a unit which binds a nucleic acid,which can selectively bind one of the nucleic acid strands (extensionproducts) produced by the amplification and can either be bound to asolid phase (immobilizable) or is bound to a solid phase (immobilized).In this connection a direct binding is understood as the binding of twobases with the aid of hydrogen bridges. Binding to a solid phase can becovalent or non-covalent. Covalent bonds can either be ensured byphosphodiester bonds (EP-B-0 386 229) and amide bonds (EP-B-0 562 025 orU.S. Pat. No. 5,242,974) or by activation of photosensitive residues(e.g. diazo groups) and contact with a solution in a probe whichcontains a reactive group (e.g. PCT/EP96/00893).

Non-covalent bonds are for example biospecific bonds such as thosecontained in the interacting pairs (strept)avidin-biotin,hapten-antibody, vitamin-receptor etc. The capture probe is preferablylabelled with biotin e.g. via a 5′ phosphate group with the aid of anaminoalkyl linker and the solid phase is coated with streptavidin.However, the capture probe is preferably already bound covalently or viaa stable biospecific bond (e.g. (strept)avidin-biotin) to the solidphase before contact with the reaction mixture from the amplification.The constitution of the solid phase is not important for the presentinvention. However, it should be insoluble in the amplification mixture.Phases made of plastics with or without a metallic component e.g. in theform of beads or a porous fleece or a more or less flat, non-porous testcarrier are for example particularly suitable. An important property ofthe carrier is that it represents a site at which the capture probes arebound or can be bound, and where the nucleic acid to be detected canbind if it is present. It is preferable not to use a capture probe whichis 100% complementary to another capture probe at the same time.

A particular embodiment of the invention is directed towards thepotential (multiple) detection of a plurality of nucleic acids withdifferent sequences. This can be achieved by using a capture probe thatis selective for each nucleic acid or sequence to be detected. Althoughthe capture probes can be bound to different solid phases, they areparticularly preferably bound to different sites and particularlypreferably to defined geometric sites on the same solid phase that canbe distinguished by the measurement technique preferably of the nucleicacid array type as described for example in WO 92/10588. In thisconnection it is possible to select certain geometric patterns such asrectangular, hexagonal or cross-shaped matrices which facilitate theevaluation. This enables the presence or absence of whole clusters oftarget sequences to be examined in a sample. The present inventionutilizes the formation of a binding complex (hybrid) which contains theextension product of the reverse as well as of the forward primer andalso the capture probe. In the following the extension product which hasbeen formed by extension of the forward primer is referred to as theforward extension product. In the methods known from the prior art therequired conditions have not been met for the measurable formation ofsuch a hybrid. In general the formation of such a complex is favouredwhen the capture probe is given sufficient opportunity to hybridize witha strand of the amplificate before this can hybridize with its(amplificate) opposite strand to form a (re)association product. If thisis not the case, the capture probe can no longer hybridize with thedesired extension product especially in the case of nucleic acids to bedetected which have a strong tendency for internal strand hybridformation and the detection is impeded or made impossible since thesignal strength is then quite small.

This can be achieved as follows: a suitable amount of hybridizationsolution and an aliquot from the reaction mixture from the amplificationin which extension products present as double-strands have beenconverted into single strands by base denaturation are each separatelyor/and successively picked up in a pipette, preferably in the samepipette, physically separated from one another (so that mixing in thepipette is essentially excluded) and then ejected into a vessel whichcontains the solid phase.

In a preferred embodiment firstly the hybridization solution, then anair bubble and then the reaction mixture are aspirated into a pipette ora needle so that the two liquids do not mix if possible. Then the twoliquids are rapidly dispensed into a vessel which contains the captureprobe i.e. within a period of less than 5 sec. preferably less than 1sec. Rapid mixture is achieved in particular by the speed of thedispensing and if desired by repeated uptake and ejection of the liquidinto and out of the pipette. In this connection the rate of dispensingmust be matched to the geometry of the reaction vessel e.g. in order toavoid splashing. Rapid mixing ensures that the hybridization of onestrand of the extension product with capture probe can take placepractically simultaneously to strand reassociation or the internalstrand hybridization. The starting time for the hybridization is denotedT₀ and is the time at which reaction conditions have been set whichallow for hybridization.

With regard to the incubation temperature it is preferably to choose oneat which the capture probe can hybridize with the sense strand,preferably ≧5° C. below the melting point of this hybrid and ≧2° C.above the freezing point of the mixture.

The probe(s) are preferably incubated with the reaction mixture forbetween 45 and 180 min. Subsequently the fraction of primers (inparticular of the labelled primer) and of labelled amplificationproducts which has not bound to the solid phase is separated from thesolid phase so that non-selectively bound i.e. excess label does notsignificantly interfere with the determination of the selectively boundfraction. This can be achieved by removing the liquid by pipette and canbe optionally facilitated by one or several wash steps.

In a preferred embodiment of the invention relatively unstringenthybridization conditions are selected for the incubation of the captureprobe with the amplificates i.e. so that selective hybridization doesnot yet occur but a relatively large amount of nucleic acid isrelatively unselectively bound. In contrast relatively stringenthybridization conditions are selected in one or several of thesubsequent wash steps. In this process, hybrids in which the captureprobe is relatively less complementary to the target sequence aredetached again whereas the more stringently complementary hybrids remainbound.

For this purpose the wash buffer is preferably chosen as follows: Itcontains 2 to 5 mol/l TMAC, 0.1-5 mmol/l of a chelating agent (e.g.EDTA), 0.1 to 5% by weight of a preferably anionic detergent and 1 to100 mM of an organic buffer base.

The present procedure is particularly suitable for detecting mutationsin particular those with a complex pattern e.g. resistances of organismsfor example towards antibiotics or polymorphisms such as p53. It ischaracterized in that it is particularly selective i.e. candifferentiate or discriminate particularly well between very similarnucleic acid sequences.

In a preferred embodiment the sample, e.g. a serum, is treated with theaid of reagents such that the nucleic acids to be detected are presentin a form that is accessible for the hybridization. This can for examplebe achieved by the addition of reagents which lyse the cell walls oforganisms e.g. viruses or bacteria. If desired the nucleic acids can beprepurified e.g. by immobilization on a solid phase e.g. glass particleswith the addition of chaotropic salts. Such prepurifications aredescribed for example in EP-A-0 389 063 or U.S. Pat. No. 5,234,809. Thesolution containing nucleic acids is then subjected to a nucleic acidamplification by PCR in which the reverse primer is labelled with adetectable group but not the forward primer. The region of the nucleicacid to be detected that is located between the primers is the regionwhich contains the target sequence. The solution containing amplificateis added at the same time as a hybridization solution to a flat reactionvessel on the surface of which capture probes with different sequencesare bound in discrete areas and the capture probes are eachcomplementary to one target sequence on the unlabelled sense strand of anucleic acid to be detected. If, in contrast, the capture probes aredesigned to be antiparallel complementary to the anti-sense strand, theforward primer is correspondingly used in a labelled form.

In this process the desired hybrids composed of the capture probe andthe two strands are formed according to the invention provided thenucleic acid to be detected was present in the original sample. Theformation of the hybrids can be detected via the label incorporated inthe amplificates e.g. directly by means of a (fluorescence) microscope.In the case of indirect labelling e.g. by a hapten, labelled antibodyspecific to the hapten is added so that the antibody and thus the(second) label binds to the (first) label. Subsequently it is possibleto measure the (second) label.

The measurement is evaluated by establishing at which positions withinthe array of specific reaction sites a label is measured. The sequencethat is complementary to the (known) sequence of the capture probe thatis bound there is then present in the sample. In order to decide whetherthe measured signal can be rated as positive it is usual to define athreshold value above which it is assumed that the nucleic acid waspresent. Since the measured signals can vary for different sequences andsimilarities, and the measurement sensitivity is usually so high thatbackground signals could also be inspected as a signal, the increasedpower of discrimination that can be achieved by the present invention isextremely important in order to avoid false assessments. It enables avery much improved differentiation between the signals in the presenceand absence of the target sequence.

In such multiple tests it is possible on the one hand to detect diverseanalytes e.g. viruses such as HBV, HGV, HCV and HIV together usingvarious primer pairs or partial sequences of a particular nucleic acide.g. polymorphisms on human genomes but it is also possible to determinesequences by the sequencing by hybridization (SBH) method either de-novo(previously unknown sequences) or to find deviations from a normalsequence. An SBH method is described for example by Khrapko et al., FEBSLetters 250, 118-122 (1989) or Khrapko et al., J. DNA Sequencing andMapping 1, 375-388 (1991). In this method capture probes ofapproximately the same length are usually used the sequences of whichoverlap such that they are shorter at one end and longer at the otherend than the sequence of nearest similarity. The same applies for eachadditional sequence which are in an unchanged direction longer at oneend and shorter at the other end. The shift that is formed in thismanner is between 1 and 5 nucleotides according to requirements. Thesequence of the nucleic acids to be sequenced can be determined bycombining the hybridization information obtained.

Allele-specific or mutation-specific capture probes can be used formutation analysis. Therefore a subject matter is also a method for theselective detection of a nucleic acid comprising the steps ofamplification of the nucleic acid or of a part thereof with the aid oftwo primers one of which can hybridize with one strand of the nucleicacid to be detected and the other can hybridize with a complementarystrand thereto and at least one of which contains a bound detectablelabel to form one extension product of these primers in a reactionmixture, binding a capture probe to one of these extension products toform a hybridization complex which contains the capture probe and atleast this extension product, separating the extension product bound tothe capture probe from non-bound components of the reaction mixture anddetermining the detectable label bound to the capture probe wherein thecapture probe is selected such that it can bind with the strand of theextension product which can also hybridize with the extension productformed by extension of the labelled primer.

A further subject matter is a reagent kit for the detection of nucleicacids containing in separate containers at least one primer whichcontains a bound detectable label for the amplification of nucleic acidsor parts thereof, at least two capture probes with different sequencescharacterized in that the primer is selected such that it can hybridizewith the same strand of the extension product as the capture probes.

An additional subject matter of the invention is the use of a reagentkit containing at least one detectably labelled primer and at least 2capture probes with different sequences wherein the at least onedetectably labelled primer is selected such that it can hybridize withthe same strand of each target nucleic acid as the capture probes in amutation analysis.

The present invention is elucidated by the following examples:

EXAMPLES Example 1

Detection Of Mycobacterium Tuberculosum

A. Sample Preparation and Amplification

DNA plasmid standards cloned in E. coli which contain the gene for the βsubunit of the RNA polymerase (rpo β) of Mycobacterium tuberculosis(wild-type WT and mutants MX) were isolated after cell lysis andpurified by means of small Quiagen columns. The rpo β gene segment thatis relevant for rifampicin resistance was amplified by PCR in whichoptionally the forward or the reverse primer carried a DIG label at the5′ end. The primer oligonucleotides were synthesized according tostandard phosphoramidite chemistry (Caruthers, M. H., Barone A. D.,Beaucage, S. L., Dodds, D. R., Fisher, E. F., McBride, L. J., Matteucci,M., Stabinsky, Z., Tang, J. Y., Chemical synthesis ofdeoxyribonucleotides, Methods in Enzymology 154, 287-313 (1987)) andoptionally 5′ terminally functionalized by incorporation of[N-trifluoro-acetamido-(3-oxa)pentyl-N,N-diisopropyl-methyl-phsophoramidite(Boehringer Mannheim Company (BM), order No. 1480863), subsequent basiccleavage of the trifluoroacetyl protective group with aqueous ammoniaand reaction withdigoxigenin-3-O-methylcarbonyl-6-amidocaproyl-NHS-ester (BM Company,order No. 1333054).

The PCR temperature profile run on a Perkin-Elmer GeneAmp PCR system9600 using 1 μl purified cell extract per 100 μl PCR reaction solutionwas:

Initiation 3′94° C.; 10 cycles 15″ 95° C./30″ 68° C./30″ 72° C.; 20cycles 15″ 94° C./30″ 68° C./30″+additionally 20″ with each new cycle at72° C.; 5′ 70° C. Equilibration≧30′ 4° C.

PCR buffer: 10 mM Tris/HCl, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3, primer(forward and reverse, respectively, see below) polymerase and dNTPsaccording to PCR Core Kit (BM, catalogue No. 1578553).

PCR primer (R = reverse; F = forward): primer 55F:5′-TCG CCG CGA TCA AGGAGT-3′ (SEQ ID NO.1) primer 55R:5′-TGC ACG TCG CGG ACC TCC A-3′ (SEQ IDNO.2) primer 56F:5′-DIG-TCG CCG CGA TCA AGG AGT-3′ (SEQ ID NO.3) primer56R:5′-DIG-TGC ACG TCG CGG ACC TCC A-3′ (SEQ ID NO.4) MTB rpo-β amplicon(sense strand; 157 nt): 5′-TCG CCG CGA TCA AGG AGT TCT TCG GCA CCA GCCAGC TGA GCC (SEQ ID NO.5) AAT TCA TGG ACC AGA ACA ACC CGC TGT CGG GGTTGA GCC ACA AGC GCC GAC TGT CGG CGC TGG GGC CCG GCG GTC TGT CAC GTG AGCGTG CCG GGC TGG AGG TCC GCG ACG TGC A-3′

Amplified plasmid standards, wild-type (WT) or mutants (MX) werealiquoted after being checked by gel electrophoresis and ethidiumbromide staining and stored deep-frozen until use.

B) Denaturation of the Amplificates

Firstly 5 μl of a solution of the DIG-labelled amplificate and 5 μl of astrongly basic denaturation solution (50 mM NaOH, 2 mM EDTA) are mixedin an inert reaction vessel (e.g. from the Eppendorf Co.) and incubatedfor ≧10 min at RT.

C) Manufacture of the Disposables

Capture probes that are antiparallel complementary to the sense ampliconstrand were also synthesized by standard phosphoramidite chemistry as 18mers with a 5′-terminal biotinylation. The biotin group was incorporateddirectly during the synthesis by means of abiotinoyl-6-amidohexyl-N,N-diisopropyl-β-cyanoethyl-phosphoramidite inwhich the reactive imidazolyl nitrogen is protected by dimethoxytrityl(Perkin-Elmer Company ABI, No. 401396).

c = complementary to the sense strand; S = sensitive (to rifampicintreatment ( = probes detecting the wild-type); R = resistant torifampicin treatment ( = probes detecting diverse mutants) cS1-18/2-BIO5′-BIO-T20-ATT GGC TCA GCT GGC TGG-3′ (Tab.4) SEQ ID NO.6 cR1a-18-BIO5′-BIO-T20-TTG GCT CGG CTG GCT GGT-3′ (Tab.4) SEQ ID NO.7 cS2-18-BIO5′-BIO-T20-GTT GTT CTG GTC CAT GAA-3′ (Tab.4) SEQ ID NO.8 cR2-18-BIO5′-BIO-T20-GTT GTT CTG GAG CAT GAA-3′ (Tab.4) SEQ ID NO.9 cS3-18-/1-BIO5′-BIO-T20-CAA CCC CGA CAG CGG GTT-3′ (Tab.1-3) SEQ ID NO.10cS3-18/2-BIO 5′-BIO-T20-TCA ACC CCG ACA GCG GGT-3′ (Tab.4) SEQ ID NO.11cS4-18-BIO 5′-BIO-T20-TCG GCG CTT GTG GGT CAA-3′ SEQ ID NO:12cR4a-18-BIO 5′-BIO-T₂₀-TCG GCG CTT GTA GGT CAA-3′ SEQ ID NO.13cR4b-18-BIO 5′-BIO-T₂₀-TCG GCG CTT GTC GGT CAA-3′ SEQ ID NO.14cdS4-18-BIO 5′-BIO-T₂₀-TCG GCG CTT GCG GGT CAA-3′ (Tab.4) SEQ ID NO.15cS5-18-BIO 5′-BIO-T₂₀-CCC CAG CGC CGA CAG TCG-3′ SEQ ID NO.16 cR5-18-BIO5′-BIO-T₂₀-CCC CAG CGC CAA CAG TCG-3′ (Tab.4) SEQ ID NO.17 MBD5′-BIO-T₂₅-DIG-Y-T-3′ (Tab.4) SEQ ID NO.18 BIO = monobiotinylation viaon-line incorporation of biotinoyl-6-amidohexyl phosphoramidite Y= on-line incorporation of 3-trifluoroacetamido-1,2-propandiole-phosphoramidite, subsequent basic deblocking, extension with6-aminocaproic acid NHS ester and labelling by reaction withDIG-3-0-carboxymethyl-NHS ester (base-stable) (see above) MBD: controloligo for the monitoring of conjugate/detector function.

Polystyrene disposables (DE-19707204.6) dyed with a black carbon pigmentwith a microwell measuring 0.7 cm (round, diameter)×0.15 cm (depth)served as disposables which were activated with athermoBSA-biotin/streptavidin layer (PCT/EP89/00195). Biotinfunctionalized capture probes were immobilized on this by means of aninkjet printing process according to EP-A-0 268 237. Each nozzle of theprinting head was filled with a capture probe solution with a differentspecificity to print and subsequently dry small circular reaction zoneswith a diameter of ca. 100 μm; (these are isolated from one another) ingeometric patterns on the test carrier and thus generate an array ofdifferent capture probes (C2a solid phase).

Inkjet Printing Solution: 5 mM Mes/ 5 mM Tris-HCl, pH 7.4, 1% (w/v)sucrose, 0.5 mg/ml BSA-Res (dialysed), probe concentration 1 μM.

D) Probe Hybridization

40 μl hybridization (=neutralization) buffer and 10 μl denatured PCRamplificate were aspirated successively and separated by an air bubbleusing a Hamilton MicroLab dispenser and subsequently dispensed underpressure in one step into the disposable so that a rapid mixing occursand there is an identical T₀ for all hybridization events. The mixturewas incubated for 90 min at room temperature (tables 1 to 3) or at 37°C. (table 4), with (tables 1 to 3) or without (table 4) shaking (E.Bühler Swip, 250 rpm, linear).

Hybridization Buffer

10 mM Tris/HCl, 4 M TMAC (tetramethylammonium chloride), 1 mM EDTA, 0.1%Tween-20 [in example 4: Zwittergent 3-12] (w/v), 0.013% oxypyrion, 0.01%methylisothiazolone, pH 6.3.

E) Wash Step

Firstly 20 sec b/f separation at 59 (61)° C. (set target temperature onthe manual DNA washing device) or 60° C. (actual temperature on thesemiautomatic incubator/shaker/washing module) with wash solution 1(flow rate 14.4 and 12 ml/min, respectively); immediately afterwardsrewash for 20 sec with low salt wash buffer at RT (wash solution 2, flowrate 12 ml/min).

Wash Solution 1 (For The Stringent Washing Step After Hybridization)

10 mM Tris/HCl, 4 M TMAC, 1 mM EDTA, 0.1% Tween-20 [in Tab.4:Zwittergent 3-12] (w/v), 0.013% oxypyrion, 0.01% methylisothiazolone, pH8.0.

Wash Solution 2 (to rewash for the bound/free separation afterhybridization, and for the bound/free separation after the conjugatereaction):

15 mM NaCl/1.5 mM Na₃ citrate/0.1% SDS solution, pH 7.0

F) Conjugate Binding

30 μl conjugate suspension (anti-DIG functionalized fluorobeads instable suspension, 0.01% solids) was pipetted into the well and themixture was incubated for 30 min at room temperature (60 min, 37° C. intable 3). Incubation for the final DIG: anti-DIG immune reaction isperformed with (E. Bühler Swip, 250 rpm, linear, tables 1 to 3) orwithout (table 4) shaking.

It was subsequently washed for 8 sec with low salt wash buffer (washsolution 2) at RT and at a flow rate of 12 ml/min.

Conjugate Suspension

MAB<DIG>(IgG)-110 nm COOH latex fluorobead suspension (prepared from BMbiochemicals beads cat. No. 1742582, used according to U.S. Pat. No.5,516,635) 0.1% by weight solids is prepared by dissolving a bottle ofconjugate lyophilisate with 200 μl redistilled water. The daily portionsare prepared by 1:10 dilution to 0.01% solids in conjugate buffer.

Conjugate Buffer

50 mM Tris/HCl, pH 8.5, 150 mM NaCl, 0.5% RPLA-IV, 10 μg/ml M-IgG-l(poly-Fab, BM, Biochemicals for the Diagnostic Industry, cat. No.1368388), 10 μg/ml M-IgG-2a (poly-Fab, BM, Biochemicals for theDiagnostic Industry, cat. No. 1866729), 0.05% (w/v) Tween-20, 0.095%NaN₃.

G) Measurement of the Bound Fluorescence and Evaluation

The detection was carried out using a microscope/CCD camera constructionfrom Leica Company (Heidelberg, GFR).

The measured results are compiled in tables 1-3 which were obtained withthe initial 5×(5 probe) array with a comparative evaluation of Famplicons (=sense strands labelled via forward primers) and R amplicons(=antisense strands labelled via reverse primers) with a manual testprocedure. The wash temperature in the stringent washing step afterhybridization, and the incubation temperature and time for the conjugatereaction were varied.

TABLE 1 5x array Characteristics: washing with wash buffer 1; 59° C. (20sec addition of wash buffer) conjugate 30 min shaking/RT Results:

The squares shaded grey represent a hybridization event with an exactlycomplementary probe.

TABLE 2 5x Array Characteristics: washing with wash buffer 1; 61° C. (20sec addition of washing buffer) 30 min shaking/RT

TABLE 3 5x array Characteristics: washing with wash buffer 1; 61° C. (20sec addition of washing buffer) 60 min shaking/37° C. Results

Table 4 shows the same for the extended 12×(12 probe) array under theconditions: 90 min hybridization at 37° C., 30 min conjugate reaction atRT, probe washing step at 60° C. for 20 sec carried out on asemi-automatic DNA incubator/shaker/washing module. At this time thetest was not optimized for the newly added regions 1 and 2 so thatprimarily the regions 4 and 5 (as in the smaller initial array) shouldbe considered. The position of the regions is shown in FIG. 3.

TABLE 4 12X Array R-amplicon Solid phase with various robes in separatespots Sample No Name cS3 cS4 cR4a cR4b cdS4 cS5 cR5 3 WT3.11

2.1% 0.5% 7.1%

0.8% 4 WT3.11

2.4% 0.3% 5.2%

1.2% 5 R1a

0.5% 0.2% 1.2%

2.1% 6 R1a

0.4% 0.2% 2.6%

2.8% 7 R2

0.4% 0.1% 0.4%

1.1% 8 R2

0.4% 0.1% 0.8%

1.5% 9 R4a 12.9%

1.5% 2.0%

0.9% 10 R4a 13.1%

3.3% 5.3%

3.1% 11 R4b 14.3% 7.9%

9.6%

2.7% 12 R4b 7.4% 4.2%

5.2%

0.8% 13 dS4 19.1% 0.5% 0.2%

1.3% 14 dS4 15.5% 0.4% 0.1%

0.6% 15 R5

2.5% 2.3% 14.2% 0.4%

16 R5

1.5% 0.7% 9.0% 1.4%

17 WTF/R-amp.

6.8% 1.2% 10.4%

3.2% 18 WTF/R-amp.

4.9% 1.1% 22.1%

1.8% 19 WT3.11

3.0% 0.3% 8.2%

1.0% 20 WT3.11

3.0% 0.6% 20.4%

0.6% F-amplicon Solid phase with various robes in separate spots SampleNo Name cS3 cS4 cR4a cR4b cdS4 cS5 cR5 3 WT3.11

4.6% 1.0% 20.3%

3.4% 4 WT3.11

7.6% 2.6% 18.2%

2.8% 5 R1a

0.5% 0.2% 1.7%

1.5% 6 R1a

0.8% 0.5% 0.9%

3.4% 7 R2

1.7% 0.1% 5.3%

3.6% 8 R2

3.4% 0.6% 2.6%

5.8% 9 R4a 53.6%

2.4% 1.4%

4.1% 10 R4a 90.4%

1.2% 5.8%

2.4% 11 R4b 29.3% 15.5%

15.3%

8.3% 12 R4b 54.7% 25.9%

16.6%

8.9% 13 dS4 50.4% 1.8% 0.7%

1.8% 14 dS4 51.0% 2.3% 0.5%

0.3% 15 R5

10.9% 12.3% 5.6% 6.2%

16 R5

1.9% 0.4% 3.6% 2.4%

17 WTF/R-amp.

5.8% 2.8% 17.8%

1.8% 18 WTF/R-amp.

5.5% 0.7% 24.6%

2.3% 19 WT3.11

6.4% 2.6% 6.4%

3.3% 20 WT3.11

4.5% 0.6% 21.9%

1.1%

In all cases it is clear that the use of R-amplicons i.e. an indirectdetection via the strand reassociation complex surprisingly enables asignificantly better discrimination to be achieved than with a directdetection of probe-bound F-amplicons.

This surprising effect applies in particular when single-strandedanalyte DNA or RNA has a strong tendency for internal strand secondarystructure formation so that spatial reorientation processes influencethe accessibility of the group to be detected. This is the case for theamplified rpo β-gene segment (see FIG. 4:2D folding proposal accordingto an algorithm of Dr. Zuker, Washington University; Institute forBiomedical Computing). In FIG. 5 an attempt was made to illustrate thesituation in which (unlike in the present invention) the capture probewould directly hybridize with the labelled strand in the case ofinternal strand reassociation (situation A) or no internal strandreassociation (situation B).

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 18 <210> SEQ ID NO 1 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<400> SEQUENCE: 1 tcgccgcgat caaggagt              #                  #                   #  18 <210> SEQ ID NO 2 <211> LENGTH: 19<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<400> SEQUENCE: 2 tgcacgtcgc ggacctcca              #                  #                   # 19 <210> SEQ ID NO 3 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate ester with digo #xigenin<400> SEQUENCE: 3 tcgccgcgat caaggagt              #                  #                   #  18 <210> SEQ ID NO 4 <211> LENGTH: 19<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate ester with digo #xigenin<400> SEQUENCE: 4 tgcacgtcgc ggacctcca              #                  #                   # 19 <210> SEQ ID NO 5 <211> LENGTH: 157<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<400> SEQUENCE: 5tcgccgcgat caaggagttc ttcggcacca gccagctgag ccaattcatg ga#ccagaaca     60acccgctgtc ggggttgacc cacaagcgcc gactgtcggc gctggggccc gg#cggtctgt    120 cacgtgagcg tgccgggctg gaggtccgcg acgtgca      #                   #     157 <210> SEQ ID NO 6 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 6 tttttttttt tttttttttt attggctcag ctggctgg      #                   #     38 <210> SEQ ID NO 7 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 7 tttttttttt tttttttttt ttggctcggc tggctggt      #                   #     38 <210> SEQ ID NO 8 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 8 tttttttttt tttttttttt gttgttctgg tccatgaa      #                   #     38 <210> SEQ ID NO 9 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 9 tttttttttt tttttttttt gttgttctgg agcatgaa      #                   #     38 <210> SEQ ID NO 10 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 10 tttttttttt tttttttttt caaccccgac agcgggtt      #                   #     38 <210> SEQ ID NO 11 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 11 tttttttttt tttttttttt tcaaccccga cagcgggt      #                   #     38 <210> SEQ ID NO 12 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 12 tttttttttt tttttttttt tcggcgcttg tgggtcaa      #                   #     38 <210> SEQ ID NO 13 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 13 tttttttttt tttttttttt tcggcgcttg taggtcaa      #                   #     38 <210> SEQ ID NO 14 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 14 tttttttttt tttttttttt tcggcgcttg tcggtcaa      #                   #     38 <210> SEQ ID NO 15 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 15 tttttttttt tttttttttt tcggcgcttg cgggtcaa      #                   #     38 <210> SEQ ID NO 16 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 16 tttttttttt tttttttttt ccccagcgcc gacagtcg      #                   #     38 <210> SEQ ID NO 17 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: Phosphate linked to bioti #n via Aminolinker<400> SEQUENCE: 17 tttttttttt tttttttttt ccccagcgcc aacagtcg      #                   #     38 <210> SEQ ID NO 18 <211> LENGTH: 27<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<220> FEATURE: <221> NAME/KEY: misc_signal <222> LOCATION: (27)..(27)<223> OTHER INFORMATION: Y means incorporation of  #Aminolinker-      phosphoramidite subsequently      estered with 3-O carboxymethyl digox #igenin <400> SEQUENCE: 18tttttttttt tttttttttt tttttyt           #                  #             27

What is claimed is:
 1. A method for the detection of a nucleic acidcomprising the steps: a) amplifying the nucleic acid or a part thereofin a reaction mixture with a first primer and a second primer, one ofwhich can hybridize with one strand of the nucleic acid to be detectedand the other of which can hybridize with a strand complementarythereto, wherein one of the primers contains a detectable label, to forma labeled extension product and an unlabeled extension product which arecomplementary to each other; b) contacting a capture probe with theextension products to form a hybridization complex wherein the complexis necessarily a duplex, said hybridization complex comprising thecapture probe, the labeled extension product and the unlabeled extensionproduct, wherein the capture probe is selected such that the captureprobe hybridizes to only the unlabeled extension product and only theunlabeled extension product hybridizes with the labeled extensionproduct; c) separating the hybridization complex from other componentsof the reaction mixture; and d) determining the detectable label in thehybridization complex of step b).
 2. The method of claim 1, wherein thecapture probe is bound to a solid phase.
 3. The method of claim 2,wherein the hybridization complex comprising the solid phase-boundcapture probe is visualized by a detection reagent that i) provides asignal or generates a signal and ii) contains a component capable ofbinding to the primer label.
 4. The method of claim 3, wherein thedetection reagent contains a particulate signal-transmitting or signalmediating component.
 5. A method for the selective detection of specificsequences in one or several nucleic acids(s) from a sample comprisingthe steps: a) amplifying the nucleic acids or parts thereof in areaction mixture with at least one set of two primers, wherein a firstprimer in each set of primers can hybridize with one strand of thenucleic acid(s) and a second primer in each set can hybridize with astrand complementary thereto, wherein one of the first and secondprimers contains a detectable label, to form a labeled extension productand an unlabeled extension product which are complementary to each otherfor each of the specific sequences; b) contacting at least one captureprobe with the extension products to form hybridization complexeswherein the complexes are necessarily duplexes, said hybridizationcomplexes comprising a capture probe, the labeled extension product andthe unlabeled extension product for each of the specific sequences,wherein the capture probe is selected such that the capture probehybridizes to only the unlabeled extension product and only theunlabeled extension product hybridizes with the labeled extensionproduct; c) separating the hybridization complexes from other componentsof the reaction mixture; and d) determining the detectable label in thehybridization complexes of step b).
 6. The method of claim 5, comprisingat least two distinct capture probes which are bound to spatiallyseparate, discrete areas of a solid phase.
 7. The method of claim 5,wherein the labeled probe is used to form extension products from aplurality of specific sequences.
 8. The method of claim 6, wherein theat least one detectably labeled primer is selected such that it canhybridize with the same strand of each target nucleic acid as thecapture probes in a mutation analysis.
 9. The method of claim 1, whereinin said contacting step b), the extension products are provided insingle-stranded form.
 10. The method of claim 9, wherein in saidcontacting step b), conditions are chosen such that hybridization forthe unlabeled extension product with the capture probe occurspractically simultaneously with hybridization of the unlabeled extensionproduct with the labeled extension product.
 11. The method of claim 9,wherein the extension products are provided in single-stranded form byconverting double-stranded extension products into single-strandedextension products by alkaline denaturaton, and the method furthercomprises the step of neutralizing the alkaline before the extensionproducts are contacted with the capture probe.